The present invention relates to the treatment of systemic fungal infections by administration of liposomeincorporated polyenes.
Clinical observations and animal experimental studies have added to the understanding of host-fungal interactions. It is becoming recognized that host defense against fungal disease is multifactorial and may vary, depending on the etiologic agent. The mechanisms of resistance are not well defined in most instances, but various innate barriers and cell-mediated immune responses seem to be of primary importance. At this time, the role of antibody in resistance is uncertain. Clearly, debilitation of innate defenses and of cell-mediated immune responses can increase an individual's susceptibility to severe fungal disease from opportunistic agents such as Cryptococcus neoformans and species of Candida and Aspergillus, as well as from fungal pathogens such as Histoplasma capsulatum and Coccidioides immitis. The difficulty in gaining a complete understanding of the critical host defenses has been further complicated by many studies that show fungi may affect various host immune functions adversely. Although it is too early to evaluate the clinical importance of many of these experimental findings, investigators have demonstrated that fungi impair neutrophil function, induce IgE responses, and cause suppression of cell-mediated immune responses.
Host changes likely to be associated with increased susceptibility may be accidentally induced, as in traumatic injuries (such as burns or puncture wounds); self-induced, as in chronic alcoholism; naturally occurring, as in diabetes mellitus, various congenital immune deficiencies, collagen diseases, lymphoreticular neoplastic disease, and other types of tumors; or iatrogenically induced by instrumentation (such as catheterization), surgical procedures (such as open heart surgery), or by use of cytotoxic drugs (as in an attempt to prevent graft rejection and to treat neoplastic disease), corticosteroid therapy, and long-term use of broad-spectrum antibodies.
Chemical factors that aid resistance to fungal diseases are poorly defined. Knowledge of these substances is based primarily on circumstantial evidence at the clinical level and in vitro observations at the experimental level. Hormonally associated increases in lipid and fatty acid content on the skin occurring at puberty have been correlated with increased resistance to tinea capitis caused by the dermatophyte Microsporum audouinii, although pubescent changes are not the sole factors in resistance. Substances in serum, cerebrospinal fluid, and saliva may limit growth of Cryptococcus neoformans, and basic peptides in body fluids have been shown to inhibit Candida albicans.
Results of clinical and experimental studies indicate that C. albicans, C. neoformans, Aspergillus fumigatus, and C. immitis activate the alternative pathway of the complement cascade. Because of the polysaccharide nature of fungal cell walls, it is expected that all medically important fungi activate complement. Such activation may be important in defense against some mycoses; a positive correlation has been demonstrated between animals deficient in late-acting complement components (C3-C9) and increased susceptibility to fungi such as C. neoformans and C. albicans. Assuming that phagocytic cells are important in resistance to fungi, complement activation may play a role by provoking an acute inflammatory response on generation of complement fragments C3a and C5a, and by coating the fungal elements with opsonic fragments C3b and C3d for ingestion by phagocytic cells.
The systemic mycoses of humans and other animals are caused by some fungi that are pathogenic and cause disease in the healthy host, and by other fungi (opportunistic pathogens) that are usually innocuous but cause disease in patients whose immune defenses are impaired. Some of these fungi may be saprophytes in nature (soil, bird droppings), whereas others are a part of the normal human flora (commensals). In no case are humans the solitary or necessary host.
An example of a soil saprophyte is Histoplasma capsulatum, which commonly causes infection in endemic areas; 80%-90% of adults react positively to histoplasmin in delayed cutaneous hypersensitivity tests. An example of an opportunistic pathogen is Candida albicans, normally present in the oral cavity, gastrointestinal tract, and probably the skin. In the patient with acute leukemia, however, C. albicans is commonly present in bloo a fulminant, usually fatal, septicemia. Other opportunistic infections are seen in patients with diabetic acidosis (mucormycosis) and Hodgkin's disease (for example, cryptococcosis and histoplasmosis). The pathogenesis of these mechanisms is obscure, but cell-mediated immunity seems to be essential for a good prognosis.
Neither active vaccines nor passive immune serum immunization has been sufficiently successful to result in commercially available preparations.
Treatment of active disease may be symptomatic (for example, pain relief), sometimes surgical (resection of irremedially damaged tissue and correction of hydrocephalus), and, most successfully, chemotherapeutic (Table 1). Among the agents commonly used are hydroxystilbamidine isethionate, amphotericin B, 5-fluorocytosine (Flucytosine), miconazole, and ketoconazole. Response to these drugs varies according to the fungus, type of disease, and course of illness. For example, response is good in most B. dermatitidis infections, but is poor in most diseases caused by A. fumigatus. Response is better for skin lesions caused by B. dermatitidis than for meningitis due to C. immitis; response is better in chronic cryptococcosis than in fulminant candidiasis. Table 1 shows a listing of some systemic mycoses and generally accepted chemotherapeutic agents.
TABLE 1 ______________________________________ CHEMOTHERAPEUTIC AGENTS FOR SYSTEMIC MYCOSES Disease First Choice Second Choice ______________________________________ Aspergillosis Amphotericin B Ketoconazole Blastomycosis Amphotericin B Hydroxystilbamidine isethionate Candidiasis Amphotericin B Flucytosine or ketoconazole Coccidioidomycosis Amphotericin B Ketoconazole Cryptococcosis Amphotericin B Either drug alone* Flucytosine Histoplasmosis Amphotericin B Ketoconazole* Mucormycosis Amphotericin B Miconazole* Paracoccidioidomycosis Amphotericin B Sulfonamides, Ketoconazole* ______________________________________ *Depending on minimal inhibitory concentration necessary for the fungus.
Infection is the cause of death in 51% of patients with lymphoma and 75% of patients with leukemia. Although bacteria are the causative organisms of many such infections, fungi account for 13% of the fatal infections in patients with lymphoma and for more than 20% of patients with leukemia. The fungus Candida albicans causes more than 80% of these infections, and Aspergillus spp. is also a frequent cause of such infections. In addition, fungal infection is a major cause of morbidity and mortality in patients with congenital and acquired deficiencies of the immune system. Much concerted effort has been expended in search of agents useful in treating fungal infections of humans. As a result, many compounds have been isolated and shown to have antifungal activity, but problems associated with solubility, stability, absorption, and toxicity have limited the therapeutic value of most of them in human infections. The most useful antifungal antibiotics fall into one of two categories: those that affect fungal cell membranes and those that are taken up by the cell and interrupt vital cellular processes such as RNA, DNA, or protein synthesis. Table 2 lists some useful antifungal agents and their mechanisms of action.
TABLE 2 ______________________________________ SOME USEFUL ANTIFUNGAL AGENTS, THEIR CHEMICAL CLASSIFICATION, AND THEIR MECHANISMS OF ACTION Class Compounds Mechanism ______________________________________ Polyene Amphotericin B Interacts with sterols Nystatin (ergosterol) in fungal Hamycin cell membrane, rendering Lucensomycin cells selectively permeable to the outflow of vital constituents, e.g. potassium Imidazole Miconazole Inhibits demethylation of Clotrimazole lanosterol thus Ketoconazole preventing formation of ergosterol, a vital component of fungal cell membrane; also has a direct cidal effect on fungal cells Pyrimidine 5-Fluorocytosine Is taken up and deaminated by susceptible cell to form 5-fluorouracil, which in turn inhibits RNA synthesis; also thought to inhibit thymidylate synthetase and DNA synthesis Grisan Griseofulvin Binds to tubulin and inhibits microtubule assembly 3-Arylpyrrole Pyrrolnitrin Appears to inhibit terminal electron transport between succinate or NADH and coenzyme Q Glutaramide Cycloheximide Inhibits protein synthesis at 80S ribosomal level, preventing transfer of aminoacyl tRNA to the ribosome ______________________________________
The polyene macrolide antibiotics are secondary metabolites produced by various species of Streptomyces. Several common features of these compounds are useful in classifying the more than 80 different polyenes that have been isolated. All are characterized by a macrolide ring, composed of 26-38 carbon atoms and containing a series of unsaturated carbon atoms and hydroxyl groups. These features of the molecule contribute to the polyenes, amphipathic properties (those relating to molecules containing groups with different properties, for example, hydrophilic and hydrophobic). The ring structure is closed by the formation of an internal ester or lactone bond (FIG. 1). The number of conjugated double bonds vary with each polyene, and the compounds are generally classified according to the degree of unsaturation.
Toxic effects of polyene macrolides appear to be dependent on binding to cell membrane sterols. Thus, they bind to membranes of fungus cells as well as to those of other eukaryotic cells (human, plant, and protozoa), but not to bacterial cell membranes, which do not contain membrane sterols. The interaction of polyene macrolides with mammalian and fungal membrane sterols results in transmembrane channels that allow the leakage of intracellular components leading to cell deaths.
The usefulness of an antibiotic is usually measured by the differential sensitivity of the pathogen and host. The polyene macrolide compounds, for example, hamycin and lucensomycin, are relatively specific for fungi and are potentially useful in humans. The relative specificity of these two polyene macrolides is based on their greater avidity for ergosterol, the principal sterol of fungal membranes, compared to cholesterol, the principal sterol of human cell membranes. However, it is the binding to cholesterol which causes the toxicities typically associated with the polyene macrolide compounds. Because the polyene macrolides are so potentially useful, researchers are actively investigating ways to reduce the toxic effects of these compounds.
It has recently been shown that the encapsulation of certain drugs in liposomes before administration to the patient can markedly alter the pharmacokinetics, tissue distribution, metabolism and therapeutic efficacy of these compounds. Liposomes may be defined as lipid vesicles which are formed spontaneously on addition of an aqueous solution to a dry lipid film. Further, the distribution and pharmacokinetics of these drugs can be modified by altering the lipid composition, size, charge and membrane fluidity of the liposome in which they are encapsulated.
Recently, liposomes have been used as carriers of Amphotericin B for treatment of murine leishmaniasis (New, R. R. C., et al., "Antileishmanial Activity of Amphotericin and Other Antifungal Agents Entrapped in Liposomes." J. Antimicrob. Chemother., Vol. 8 (1981), pp. 371-381), histoplasmosis (Taylor, R. L., et al., "Amphotericin B in Liposomes: A Novel Therapy for histoplasmosis." Am. Rev. Respir. Dis., Vol. 125 (1982), pp. 610-611), cryptococosis (Graybill, J. R., et al., "Treatment of Murine Cryptococosis with Liposome-Associated Amphotericin B." J. Infect. Dis., Vol. 145 (1982), pp. 748-752). and candidiasis (Tremblay, C., et al., "Comparative Efficacy of Amphotericin B (AMB) and Liposomal AMB (lip-AMB) in Systemic Candidiasis in Mice." Abstr. 1983 ICAAC, No. 755 (1983), p. 222). Liposome-encapsulated Amphotericin B has also been used for treatment of coccidioidomycosis in the Japanese macaque (Graybill, J. R., et al., "Treatment of Coccidioidomydosis (cocci) in Primates Using Liposome Associated Amphotericin B (Lipo-AMB)." Abstr. 1982 ICCAC, No. 492 (1982), p. 152).
The present inventors have recently demonstrated that liposome encapsulated amphotericin B (AmpB) may be used to treat experimental murine candidiasis (Lopez-Berestein et al., J. Infect. Dis., Vol. 120, pp 278-283 (1984) and in the treatment of fungal infections in patients with leukemia and lymphoma (Lopez-Berestein et al., J. Infect. Dis., Vol. 151, pp 704-71- (1985).
Liposome encapsulation has markedly reduced the toxicity and enhanced the therapeutic index of polyene macrolide compounds. However, liposome formulations presently available do not sufficiently reduce toxicity of several polyene macrolide compounds, for example, hamycin, lucensomycin, and mepartricin. Accordingly, these drugs are not widely available as therapeutic agents. Recently, the present inventors have discovered that by increasing the percentage of cholesterol in the liposome formulation, the toxicity typically associated with polyene macrolide compounds is greatly reduced. Thus, the present inventors have demonstrated that liposome formulations having cholesterol concentrations as high as 60% by weight increased the rate of survival in mice treated with polyene macrolide compounds threefold. In these studies, the present inventors demonstrated that liposomes containing a large percentage by weight of cholesterol, reduced the toxicity of polyene macrolide compounds so that they may now be used relatively safely.